This invention relates to methods and devices that use microvolume compartments to effect rapid and accurate detection and enumeration of microorganisms.
The detection and enumeration of microorganisms is practiced in numerous settings, including the food-processing industry (testing for the contamination of food by microorganisms such as E. coli and S. aureus), the health care industry (testing of patient samples and other clinical samples for infection or contamination), environmental testing industry, pharmaceutical industry, and cosmetic industry.
Growth-based detection and enumeration of microorganisms is commonly practiced using either liquid nutrient media (most probable number analysis (MPN)) or semi-solid nutrient media (direct counting using, e.g., agar petri dishes). Enumeration using the liquid MPN method is typically achieved by placing serial 10-fold dilutions of a sample of interest in replicate sets of tubes containing selective media and chemical indicators. The tubes are incubated at elevated temperature (24-48 hours) followed by examination for organism growth. A statistical formula, based on the number of positive and negative tubes for each set, is used to estimate the number of organisms present in the initial sample.
This method of performing MPN analysis has several disadvantages. It is labor intensive because of the multiple diluting and pipetting steps necessary to perform the analysis. In addition, it is only practical to use replicate sets of about three to five tubes for each dilution. As a result, the 95% confidence limits for an MPN estimate for microbial concentration are extremely wide. For example, a three tube MPN estimate of 20 has 95% confidence limits ranging from 7 to 89.
In contrast to the method described above, a direct count of viable microorganisms in a sample can be achieved by spreading the sample over a defined area using nutrient media containing a gelling agent. The gelling agent (agar) prevents diffusion of the organisms during incubation (24-48 hours), producing a colony in the area where the original organism was deposited. There is, however, a limit to the number of colonies that can fit on a given area of nutrient media before fusion with neighboring colonies affects the accuracy of the count. This usually necessitates performing several dilutions for each sample. In addition, the classes of chemical indicator molecules that can be used for identifying individual types of microorganisms present within a mixed population are limited to those that produce a product that is insoluble in the gelled media.
In addition to these disadvantages, both the currently used MPN analysis and gel-based systems require a relatively long incubation time before a positive result can be detected.
The method of the present invention solves the problems associated with currently used systems. In general, this invention provides a method to effect rapid and accurate detection and enumeration of microorganisms based on the surprising result that the use of microvolumes substantially increases the speed of detection. As used herein, the term xe2x80x9cmicroorganismxe2x80x9d includes all microscopic living organisms and cells, including without limitation bacteria, mycoplasmas, rickettsias, spirochetes, yeasts, molds, protozoans, as well as microscopic forms of eukaryotic cells, for example single cells (cultured or derived directly from a tissue or organ) or small clumps of cells. Microorganisms are detected and/or enumerated not only when whole cells are detected directly, but also when such cells are detected indirectly, such as through detection or quantitation of cell fragments, cell-derived biological molecules, or cell by-products.
In one aspect, the invention features a method for detecting a microorganism in a liquid test sample. The method involves the steps of:
distributing microvolumes of the sample to a plurality of microcompartments of a culture device; incubating the culture device for a time sufficient to permit at least one cell division cycle of the microorganism; and detecting the presence or absence of the microorganism in the microcompartments.
As used herein, the term microvolume refers to a volume of between about 0.01 and about 25 microliters, and the term xe2x80x9cmicrocompartmentxe2x80x9d refers to a compartment having a capacity, or volume, to hold a microvolume of liquid test sample.
In preferred embodiments, the method further includes the step of quantitating the microorganisms in the liquid test sample. The quantitation may include the steps of determining MPN in the sample, or it may involve enumerating the microorganisms in each microcompartment of the culture device.
In other embodiments, the microcompartments may contain a coating of nutrient medium, and the nutrient medium may further include at least one indicator substance. Alternatively, the liquid test sample may include at least one indicator substance. In either case, the indicator substance may be any indicator substance capable of providing a detectable signal in the liquid test sample. Such indicators include, but are not limited to, chromogenic indicators, fluorescent indicators, luminescent indicators, and electrochemical indicators. For purposes of this application, the term xe2x80x9celectrochemicalxe2x80x9d means a chemical indicator that changes the resistance or conductance of the sample upon reaction with a microorganism.
In another aspect, the invention features a method for detecting a microorganism in a liquid test sample. This method involves the steps of:
distributing aliquots of the sample to a plurality of microcompartments of a culture device, wherein the culture device contains a plurality of sets of microcompartments, each set having microcompartments of uniform size and the sets varying in microcompartment size; incubating the culture device for a time sufficient to permit at least one cell division cycle of the microorganism; and detecting the presence or absence of the microorganism in the microcompartments.
In preferred embodiments, the microcompartments of these methods are of uniform size and each microcompartment has a volume of about 0.01 to about 25 microliters. More preferably, each microcompartment has a volume of about 0.1 to about 10 microliters, and even more preferably, of about 1 to about 2 microliters.
The culture device preferably contains 1 to about 100,000 microcompartments, more preferably about 100 to about 10,000 microcompartments, even more preferably about 200 to about 5,000 microcompartments, and most preferably about 400 to about 600 microcompartments.
In another aspect, the invention features an assay device. The device includes a substrate having a plurality of microcompartments therein, each microcompartment having a top and a bottom. The substrate may include a hydrophobic xe2x80x9cland areaxe2x80x9d between the microcompartments. Preferably the microcompartments include assay reagents, for example nutrients, gelling agents or indicator substances such as chromogenic indicators, fluorescent indicators, luminescent indicators, or electrochemical indicators. To prevent formation of air bubbles when the liquid sample is loaded into the wells, some of the microcompartments may have openings at both their tops and bottoms. The bottom surface openings are occluded by a material that is permeable to air but substantially non-permeable to aqueous liquids.
In yet another aspect, the device contains microcompartments in the form of microchannels. The microchannels may be contained on a single layer or multilayer substrate, such as a film. The device may or may not have a hydrophobic land area between the microchannels. The microchannels may comprise elongate holes that are formed in the substrate. In a preferred embodiment, the microchannels are covered with a film.
The microcompartment microchannels may comprise capillary tubes. Discrete capillary tubes may be formed/bonded together to form a device. The microchannels preferably have at least one assay reagent coated thereon.
The microcompartments can be arranged in substantially parallel rows. Typically, the volumes of the microcompartments in each row are uniform. Alternatively, the microcompartments can be arranged in various groupings or patterns for easier recognition and counting of positive signals.
The volumes of the microcompartments may range from about 0.01 to about 25 microliters, more preferably from about 0.1 to about 10 microliters, and most preferably from about 1 to about 2 microliters.
As described herein, the present invention has several advantages. First, use of microvolumes in microcompartments allows for a surprisingly rapid detection of a microorganism in a liquid test sample. Second, this rapid detection allows for rapid enumeration or quantitation of microorganisms in the liquid test sample. The invention is particularly useful in MPN analysis of a liquid test sample for a particular microorganism, such as E. coli or S. aureus. The invention allows MPN analysis to be conducted conveniently in a single device, as opposed to separate tubes, and advantageously requires a substantively shorter incubation time to reach detectable microorganism growth. Third, the use of microvolumes in microcompartments allows for the separation of a liquid test sample into a relatively larger number of test volumes. In general, the use of microvolumes in microcompartments provides a far greater number of runs, or repetitions, of a test on the liquid sample. In the case of MPN analysis, use of microvolumes in microcompartments provides a greater number of data points from which the MPN can be calculated, thereby significantly narrowing the 95% confidence limits for a given MPN result. Fourth, separation of sample into a large number of test volumes allows a higher concentration of microorganisms to be enumerated, thereby reducing or eliminating sample dilutions. Fifth, this invention allows MPN analysis to be conducted in a single device having the indicators and/or nutrients directly coated thereon. Sixth, this invention permits a wide counting range when performing MPN analysis. Other advantages of the invention will be apparent from the following description and the figures.